Oxidizing enzymes in the manufacture of paper materials

ABSTRACT

The use of fatty acid oxidizing enzymes in the manufacture of paper materials, such as paper, linerboard, corrugated paperboard, tissue, towels, corrugated containers and boxes. Examples of fatty acid oxidizing enzymes are oxygenases classified as EC 1.13.11. including any of the sub-classes thereof, such as lipoxygenase, EC 1.13.11.12. The effect of these enzymes is that the deposition of pitch is reduced, and bleaching and de-inking effects are also observed on the paper pulp and the resulting paper material. The fatty acid oxidizing enzyme can be used in combination with a substrate, with proteases, lipases, xylanases, cutinases, oxidoreductases, cellulases, endoglucanases amylases, mannanases, steryl esterases, and/or cholesterol esterases; or with surfactants and other adjuvants.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims, under 35 U.S.C. 119, the benefit ofDanish application no. PA 2001 01551, filed Oct. 23, 2001, and U.S.provisional application No. 60/336,894, filed Oct. 29, 2001, thecontents of which are fully incorporated herein by reference.

TECHNICAL FIELD

[0002] The present invention relates to the use of a fatty acidoxidizing enzyme in the manufacture of a paper material, as well as aprocess for the manufacture of a paper material, the process comprisinga step in which papermaking pulp and/or papermaking process water istreated with a fatty acid oxidizing enzyme.

BACKGROUND ART

[0003] It is well-known to use enzymes in the manufacture of papermaterials. Examples of enzymes used for this purpose are proteases,lipases, xylanases, amylases, cellulases, as well as various oxidizingenzymes such as oxidoreductases (phenol oxidizing enzymes), for examplelaccases and peroxidases.

[0004] The effects of these enzymes are wide-spread, e.g. control ofvarious deposits such as pitch, strength-improvement, de-inking,drainage improvement, tissue softening, bleaching etc.

[0005] In a papermaking process, dissolved and colloidal substances(DCS) are dispersed into the process water during the pulp and paperproduction. The DCS are often referred to as wood pitch or wood resin.Pitch causes problems in paper machines by sticking to the rollers andcausing spots or holes in the paper material.

[0006] Wood contains about 1 to 10% of pitch or extractives in additionto its main components cellulose, hemicellulose and lignin. Majorcomponents of pitch are fatty acids, triglycerides, sterols, sterylesters and so-called resin acids, e.g. abietic acid.

[0007] WO 00/53843 discloses certain steryl esterase enzyme preparationsand their use in the manufacture of paper to hydrolyze the steryl esterpart of pitch.

[0008] U.S. Pat. No. 6,066,486 discloses an enzyme preparationcomprising a cholesterol esterase derived from Pseudomonas fragi, andthe use thereof to hydrolyze pulp resin.

[0009] JP 2000080581 discloses the use of certain peroxidases for thedecomposition of abietic acid during pulping or paper making processes.

[0010] X. Zhang; Pulp & Paper Canada, 101:3 (2000), page 59-62,discloses studies of the ability of e.g. laccase to remove dissolved andcolloidal substances.

[0011] Also Karlsson et al.: Reactivity of Trametes laccases with fattyand resin acids; Appl. Microbiol. Biotechnol. (2001) 55:317-320discloses experiments in which laccases were used to treat a model pitchpreparation.

[0012] However, none of the references cited above disclose the use of afatty acid oxidizing enzyme as defined herein for the manufacture of apaper material.

SUMMARY OF THE INVENTION

[0013] The present inventors surprisingly found that certain oxidizingenzymes, viz. fatty acid oxidizing enzymes, are advantageous in themanufacture of paper materials. An important effect of these enzymes isthat the deposition of pitch is reduced. Furthermore, these enzymes havea bleaching effect on the paper pulp and the resulting paper material.And finally, a de-inking effect has been observed, too.

DETAILED DESCRIPTION OF THE INVENTION

[0014] Paper and Pulp

[0015] By the term a “paper-making process” is meant a process, whereinthe pulp is suspended in water, mixed with various additives and thenpassed to equipment in which the paper, cardboard, tissue, towel etc. isformed, pressed and dried.

[0016] The term “paper material” refers to products, which can be madeout of pulp, such as paper, linerboard, corrugated paperboard, tissue,towels, corrugated containers or boxes.

[0017] The term “a papermaking pulp” or “pulp” means any pulp which canbe used for the production of a paper material. For example, the pulpcan be supplied as a virgin pulp, or can be derived from a recycledsource. The papermaking pulp may be a wood pulp, a non-wood pulp or apulp made from waste paper. A wood pulp may be made from softwood suchas pine, redwood, fir, spruce, cedar and hemlock or from hardwood suchas maple, alder, birch, hickory, beech, aspen, acacia and eucalyptus. Anon-wood pulp may be made, e.g., from bagasse, bamboo, cotton or kenaf.A waste paper pulp may be made by re-pulping waste paper such asnewspaper, mixed office waste, computer print-out, white ledger,magazines, milk cartons, paper cups etc.

[0018] In a particular embodiment, the papermaking pulp to be treatedcomprises both hardwood pulp and softwood pulp.

[0019] The wood pulp to be treated may be mechanical pulp (such asground wood pulp, GP), chemical pulp (such as Kraft pulp or sulfitepulp), semichemical pulp (SCP), thermomechanical pulp (TMP),chemithermomechanical pulp (CTMP), or bleached chemithermomechanicalpulp (BCTMP).

[0020] Mechanical pulp is manufactured by the grinding and refiningmethods, wherein the raw material is subjected to periodical pressureimpulses. TMP is thermomechanical pulp, GW is groundwood pulp, PGW ispressurized groundwood pulp, RMP is refiner mechanical pulp, PRMP ispressurized refiner mechanical pulp and CTMP is chemithermimechanicalpulp.

[0021] Chemical pulp is manufactured by alkaline cooking whereby most ofthe lignin and hemicellulose components are removed. In Kraft pulping orsulphate cooking sodium sulphide or sodium hydroxide are used asprincipal cooking chemicals. In these types of pulp, as a result of thealkaline cooking, the triglyceride part of pitch will be hydrolysed intofatty acids and glycerol. Fatty acid oxidizing enzymes are particularlyuseful in the treatment of such pulps, because, as the designationtells, these enzymes will catalyze the further degradation of the fattyacids resulting from the alkaline hydrolysis of the triglycerides.

[0022] The Kraft pulp to be treated may be a bleached Kraft pulp, whichmay consist of softwood bleached Kraft (SWBK, also called NBKP (NadelHolz Bleached Kraft Pulp)), hardwood bleached Kraft (HWBK, also calledLBKP (Laub Holz Bleached Kraft Pulp and)) or a mixture of these.

[0023] The pulp to be used in the process of the invention is asuspension of mechanical or chemical pulp or a combination thereof. Forexample, the pulp to be used in the process of the invention maycomprise 0%, 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%,80-90%, or 90-100% of chemical pulp. In a particular embodiment, achemical pulp forms part of the pulp being used for manufacturing thepaper material. In the present context, the expression “forms part of”means that in the pulp to be used in the process of the invention, thepercentage of chemical pulp lies within the range of 1-99%. Inparticular embodiments, the percentage of chemical pulp lies within therange of 2-98%, 3-97%, 4-96%, 5-95%, 6-94%, 7-93%, 8-92%, 9-91%, 10-90%,15-85%, 20-80%, 25-75%, 30-70%, 40-60%, or 45-55%.

[0024] In a particular embodiment of the use and the process of theinvention, the chemical pulp is a Kraft pulp, a sulfite pulp, asemichemical pulp (SCP), a thermomechanical pulp (TMP), achemithermomechanical pulp (CTMP), a bleached chemithermomechanical pulp(BCTMP). In particular embodiments the Kraft pulp is bleached Kraftpulp, for example softwood bleached Kraft (SWBK, also called NBKP (NadelHolz Bleached Kraft Pulp)), hardwood bleached Kraft (HWBK, also calledLBKP (Laub Holz Bleached Kraft Pulp and)) or a mixture thereof.

[0025] Process Conditions

[0026] The process of the invention is particularly applicable to theoxidation and hydrolysis of compounds constituting the pitch during apulping or paper-making process, e.g. to avoid pitch troubles.

[0027] The process of the invention may be applied to anypitch-containing pulp, especially to pulps with a considerable contentof linoleic acid or other unsaturated free fatty acids.

[0028] In the case of paper and pulp processing, the process accordingto the invention can be carried out at any pulp production stage. Theenzyme can be added to any holding tank, e.g. to a pulp storingcontainer (storage chest), storage tower, mixing chest or meteringchest. The enzyme treatment can be performed before the bleaching ofpulp, in connection with the pulp bleaching process or after thebleaching. When carried out in connection with pulp bleaching the enzymepreparation may be added together with bleaching chemicals such aschlorine, chlorine dioxide. Applying oxygen gas, hydrogen peroxide orozone or combinations thereof may also carry out the bleaching of pulp.The enzyme preparation may also be added together with these substances.Preferably the enzyme preparation is added prior to bleaching. Theenzyme can also be added to the circulated process water (white water)originating from bleaching and process water (brown water) originatingfrom the mechanical or chemimechanical pulping process. In a particularembodiment of a Kraft pulping process, the enzyme is added during thebrown-stock washing.

[0029] In the present context, the term “process water” comprisesi.a. 1) water added as a raw material to the paper manufacturingprocess; 2) intermediate water products resulting from any step of theprocess for manufacturing the paper material; as well as 3) waste wateras an output or by-product of the process. In a particular embodiment,the process water is, has been, is being, or is intended for beingcirculated (re-circulated), i.e. re-used in another step of the process.The term “water” in turn means any aqueous medium, solution, suspension,e.g. ordinary tap water, and tap water in admixture with variousadditives and adjuvants commonly used in paper manufacturing processes.In a particular embodiment the process water has a low content of solid(dry) matter, e.g. below 20%, 18%, 16%, 14%, 12%, 10%, 8%, 7%, 6%, 5%,4%, 3%, 20% or below 1% dry matter.

[0030] The use and process of the invention does not include the use ofthe lipoxygenase derived from Magnaporthe salvinii as described inExample 2 of PCT/DK02/00251 for bleaching dye for pulp industry in wastewater.

[0031] The process of the invention may be carried out at conventionalconditions in the paper and pulp processing. The process conditions willbe a function of the enzyme(s) applied, the reaction time and theconditions given.

[0032] The enzyme of the invention should be added in an effectiveamount. By the term “effective amount” is meant the amount sufficient toachieve the desired and expected effect, such as oxidizing pitchcomponents, obtaining a desired bleaching and/or de-inking etc.

[0033] In a particular embodiment, the dosage of the fatty acidoxidizing enzyme and additional enzymes, if any, is from about 0.1 mgenzyme protein to about 100.000 mg enzyme protein (of each enzyme) perton of paper pulp.

[0034] In further particular embodiments, the amount of the fatty acidoxidizing enzyme and additional enzymes, if any, is in the range of0.00001-20; or 0.0001-20 mg of enzyme (calculated as pure enzymeprotein) per gram (dry weight) of lignocellulosic material, such as0.0001-10 mg/g, 0.0001-1 mg/g, 0.001-1 mg/g, 0.001-0.1, or 0.01-0.1 mgof enzyme per gram of lignocellulosic material. Again, these amountsrefer to the amount of each enzyme.

[0035] The enzymatic treatment can be done at conventional consistency,e.g. 0.5-10 % dry substance. In particular embodiments, the consistencyis within the range of 0.5-45; 0.5-40; 0.5-35; 0.5-30; 0.5-25; 0.5-20;0.5-15; 0.5-10; 0.5-8; 0.5-6; or 0.5-5% dry substance.

[0036] The enzymatic treatment may be carried out at a temperature offrom about 10 to about 100° C. Further examples of temperature ranges(all “from about” and “to about”) are the following: 20-100, 30-100,35-100, 37-100, 40-100, 50-100, 60-100, 70-100, 10-90, 10-80, 10-70,10-60, and 30-60° C., as well as any combination of the upper and lowervalues here indicated. A typical temperature is from about 20 to 90° C.,or 20 to 95° C., preferably from about 40 to 70° C., or 40 to 75° C.

[0037] The enzymatic treatment may be carried out at a pH of from about2 to about 12. Further examples of pH ranges (all “from about” and “toabout”) are the following: 3-12, 4-12, 5-12, 6-12, 7-12, 8-12, 9-12,2-11, 2-10, 2-9, 2-8, 4-10, 5-8 as well as any combination of the upperand lower values here indicated. A typical pH range is from about 2 to11, preferably within the range from about 4 to 9.5, or 6 to 9.

[0038] A suitable duration of the enzymatic treatment may be in therange from a few seconds to several hours, e.g. from about 30 seconds toabout 48 hours, or from about 1 minute to about 24 hours, or from about1 minute to about 18 hours, or from about 1 minute to about 12 hours, orfrom about 1 minute to 5 hours, or from about 1 minute to about 2 hours,or from about 1 minute to about 1 hour, or from about 1 minute to about30 minutes. A typical reaction time is from about 10 minutes to 3 hours,10 minutes to 10 hours, preferably 15 minutes to 1 hour, or 15 minutesto 2 hours.

[0039] Molecular oxygen from the atmosphere will usually be present insufficient quantity, if required. Therefore, the reaction mayconveniently be carried out in an open reactor, i.e. at atmosphericpressure.

[0040] Various additives over and above the fatty acid oxidizing enzymeand additional enzymes, if any, can be used in the process or use of theinvention. Surfactants and/or dispersants are often present in, and/oradded to a papermaking pulp. Thus the process and use of the presentinvention may be carried out in the presence of an anionic, non-ionic,cationic and/or zwitterionic surfactant and/or dispersant conventionallyused in a papermaking pulp. Examples of anionic surfactants arecarboxylates, sulphates, sulphonates or phosphates of alkyl, substitutedalkyl or aryl. Fatty acids are examples of alkyl-carboxylates. Examplesof non-ionic surfactants are polyoxyethylene compounds, such as alcoholethoxylates, propoxylates or mixed ethoxy-/propoxylates, poly-glycerolsand other polyols, as well as certain block-copolymers. Examples ofcationic surfactants are water-soluble cationic polymers, such asquartenary ammonium sulphates and certain amines, e.g.epichlorohydrin/dimethylamine polymers (EPI-DMA) and cross-linkedsolutions thereof, polydiallyl dimethyl ammonium chloride (DADMAC),DADMAC/Acrylamide co-polymers, and ionene polymers, such as thosedisclosed in U.S. Pat. Nos. 5,681,862; and 5,575,993. Examples ofzwitterionic or amphoteric surfactants are betains, glycinates, aminopropionates, imino propionates and various imidazolin-derivatives. Alsothe polymers disclosed in U.S. Pat. No. 5,256,252 may be used.

[0041] Also according to the invention, surfactants such as the above,including any combination thereof may be used in a paper making processtogether with a fatty acid oxidizing enzyme as defined herein, andincluded in a composition together with such enzyme. The amount of eachsurfactant in such composition may amount to from about 8 to about 40%(w/w) of the composition. In particular embodiments the amount of eachsurfactant is from about 10 to about 38, or from about 12 to about 36,or from about 14 to about 34, or from about 16 to about 34, or fromabout 18 to about 34, or from about 20 to about 34, or from about 22 toabout 34, or from about 24 to about 34, or from about 26 to about 34, orfrom about 28 to about 32% (w/w).

[0042] In another particular embodiment, each of the above ranges refersto the total amount of surfactants.

[0043] Enzymes

[0044] EC-numbers may be used for classification of enzymes, e.g. lipaseEC-number for enzymes having lipase activity, etc. Reference is made tothe Recommendations (1992) of the Nomenclature Committee of theInternational Union of Biochemistry and Molecular Biology, AcademicPress Inc., 1992.

[0045] It is to be understood that the term enzyme, as well as thevarious enzymes and enzyme classes mentioned herein, encompass wild-typeenzymes, as well as any variant thereof that retains the activity inquestion. Such variants may be produced by recombinant techniques. Thewild-type enzymes may also be produced by recombinant techniques, or byisolation and purification from the natural source.

[0046] In a particular embodiment the enzyme in question iswell-defined, meaning that only one major enzyme component is present.This can be inferred e.g. by fractionation on an appropriateSize-exclusion column. Such well-defined, or purified, or highlypurified, enzyme can be obtained as is known in the art and/or describedin publications relating to the specific enzyme in question.

[0047] Fatty Acid Oxidizing Enzyme

[0048] The term “a” fatty acid oxidizing enzyme means at least one ofsuch enzymes. The term “at least one” means one, two, three, four, five,six or even more of such enzymes.

[0049] In the present context, a fatty acid oxidizing enzyme is anenzyme which hydrolyzes the substrate linoleic acid more efficientlythan the substrate syringaldazine. “More efficiently” means with ahigher reaction rate. This can be tested using the method described inExample 2, and calculating the difference between (1) absorbancyincrease per minute on the substrate linoleic acid (absorbancy at 234nm), and (2) absorbancy increase per minute on the substratesyringaldazine (absorbancy at 530 nm), i.e. by calculating the ReactionRate Difference (RRD)=(d(A₂₃₄)/dt−d(A₅₃₀)/dt). If the RRD is above zero,the enzyme in question qualifies as a fatty acid oxidizing enzyme asdefined herein. If the RRD is zero, or below zero the enzyme in questionis not a fatty acid oxidizing enzyme.

[0050] In particular embodiments, the RRD is at least 0.05, 0.10, 0.15,0.20, or at least 0.25 absorbancy units/minute.

[0051] In a particular embodiment of the method of Example 2, theenzymes are well-defined. Still further, for the method of Example 2 theenzyme dosage is adjusted so as to obtain a maximum absorbancy increaseper minute at 234 nm, or at 530 nm. In particular embodiments, themaximum absorbancy increase is within the range of 0.05-0.50; 0.07-0.4;0.08-0.3; 0.09-0.2; or 0.10-0.25 absorbancy units pr. min. The enzymedosage may for example be in the range of 0.01-20; 0.05-15; or 0.10-10mg enzyme protein per ml.

[0052] In the alternative, a “fatty acid oxidizing enzyme” may bedefined as an enzyme capable of oxidizing unsaturated fatty acids moreefficiently than syringaldazine. The activity of the enzyme could becompared in a standard oximeter setup as described in Example 1 of thepresent application at pH 6 and 30° C. including either syringaldazineor linoleic acid as substrates.

[0053] In a particular embodiment, the fatty acid oxidizing enzyme isdefined as an enzyme classified as EC 1.11.1.3, or as EC 1.13.11.—. EC1.13.11.—means any of the sub-classes thereof, presently forty-nine: EC1.13.11.1-EC 1.13.11.49. EC 1.11.1.3 is designated fatty acidperoxidase, and EC 1.13.11.—is designated oxygenases acting on singledonors with incorporation of two atoms of oxygen.

[0054] In a further particular embodiment, the EC 1.13.11.—enzyme isclassified as EC 1.13.11.12, EC 1.13.11.31, EC 1.13.11.33, EC1.13.11.34, EC 1.13.11.40, EC 1.13.11.44 or EC 1.13.11.45, designatedlipoxygenase, arachidonate 12-lipoxygenase, arachidonate15-lipoxygenase, arachidonate 5-lipoxygenase, arachidonate8-lipoxygenase, linoleate diol synthase, and linoleate 11 -lipoxygenase,respectively).

[0055] In a further particular embodiment, the fatty acid oxidizingenzyme is a lipoxygenase (LOX), classified as EC 1.13.11.12, which is anenzyme that catalyzes the oxygenation of polyunsaturated fatty acids,especially cis,cis-1,4-dienes, e.g. linoleic acid and produces ahydroperoxide. But also other substrates may be oxidized, e.g.monounsaturated fatty acids.

[0056] Microbial lipoxygenases can be derived from, e.g., Saccharomycescerevisiae, Thermoactinomyces vulgaris, Fusarium oxysporum, Fusariumproliferatum, Thermomyces lanuginosus, Pyricularia oryzae, and strainsof Geotrichum. The preparation of a lipoxygenase derived fromGaeumannomyces graminis is described in Examples 3-4 of WO 02/20730. Theexpression in Aspergillus oryzae of a lipoxygenase derived fromMagnaporthe salvinii is described in Example 2 of PCT/DK02/00251, andthis enzyme can be purified using standard methods, e.g. as described inExample 4 of WO 02/20730.

[0057] Lipoxygenase (LOX) may also be extracted from plant seeds, suchas soybean, pea, chickpea, and kidney bean. Alternatively, lipoxygenasemay be obtained from mammalian cells, e.g. rabbit reticulocytes.

[0058] Lipoxygenase activity may be determined spectrophotometrically at25° C. by monitoring the formation of hydroperoxides. For the standardanalysis, 10 micro liters enzyme was added to a 1 ml quartz cuvettecontaining 980 micro liter 25 mM sodium phosphate buffer (pH 7.0) and 10micro liter of substrate solution (10 mM linoleic acid dispersed with0.2%(v/v) Tween 20 (should not be kept for extended time periods)). Theenzyme was typically diluted sufficiently to ensure a turn-over ofmaximally 10% of the added substrate within the first minute. Theabsorbance at 234 nm was followed and the rate was estimated from thelinear part of the curve. The cis-trans-conjugated hydro(pero)xy fattyacids were assumed to have a molecular extinction coefficient of 23,000M⁻¹ cm⁻¹.

[0059] The fatty acid oxidizing enzyme may also be applied together witha substrate for the enzyme capable of enhancing the enzymatic effect.Suitable substrates are hydrolyzed oils such as oils from soybeans (richin linoleic acid) or tall oil. Fatty acid substrates may be releasedfrom the added oil by lipolytic enzymes or produced during the Kraftpulping or sulphate cooking.

[0060] In particular embodiments the substrate is a compound with1,4-pentadien structure, e.g. with cis,cis-1,4-pentadien structure, i.e.compounds having at least one such element in its structural formula.Examples of such substrates are unsaturated fatty acids, e.g.palmitoleic acid, oleic acid, linoleic acid, linolenic acid, andarachidonic acid, as well as their salts and esters, e.g. methyl- andethyl-esters.

[0061] In further particular embodiments the substrate is linoleic acid;linoleic acid methyl or ethyl ester; linolenic acid, or linolenic acidmethyl or ethyl ester.

[0062] To explore the effect of adding a substrate for the fatty acidoxidizing enzyme in question, the following method may be used: Thespectrum of 10 mM abietic acid (emulsified in 0.2% Tween 20) isrecorded. Characteristic peaks are observed around 200 nm and around 250nm. In a first experiment, a fatty acid oxidizing enzyme is added to theabietic acid emulsion. In a second experiment, a substrate for the fattyacid oxidizing enzyme is also added. The enzyme is e.g. a lipoxygenasederived from M. salvinii as described above, and the substrate is e.g.linoleic acid. The degradation of abietic acid is followedspectrophotometrically, and the peaks around 200 nm and around 250 nmdecrease more rapidly when linoleic acid is added together with thelipoxygenase.

[0063] In particular embodiments of the above method, and of the processof the invention, the substrate, e.g. linoleic acid, is added in anamount of 5-10000 ppm (mg/l), or 10-9000, 10-8000, 25-7500, 30-7000,50-6000, 50-5000, 50-4000, 75-3000, 75-2500, 80-2000, 90-1500, 100-1000,150-800, or 200-700 ppm. In Example 4, 333 ppm of linoleic acid was usedtogether with a fatty acid oxidizing enzyme.

[0064] In further particular embodiments of the above method, and of theprocess of the invention, the fatty acid oxidizing enzyme is used in anamount of 0.005-50 ppm (mg/l), or 0.01-40, 0.02-30, 0.03-25, 0.04-20,0.05-15, 0.05-10, 0.05-5, 0.05-1, 0.05-0.8, 0.05-0.6, or 0.1-0.5 ppm.The amount of enzyme refers to mg of a well-defined enzyme preparation.

[0065] In the process of the invention, the fatty acid oxidizing enzymemay be applied alone or together with an additional enzyme. The term “anadditional enzyme” means at least one additional enzyme, e.g. one, two,three, four, five, six, seven, eight, nine, ten or even more additionalenzymes.

[0066] The term “applied together with” (or “used together with”) meansthat the additional enzyme may be applied in the same, or in anotherstep of the process of the invention. The other process step may beupstream or downstream in the paper manufacturing process, as comparedto the step in which the papermaking pulp or process water is treatedwith a fatty acid oxidizing enzyme.

[0067] In particular embodiments the additional enzyme is an enzymewhich has protease, lipase, xylanase, cutinase, oxidoreductase,cellulase, endoglucanase, amylase, mannanase, steryl esterase, and/orcholesterol esterase activity. Examples of oxidoreductase enzymes areenzymes with laccase, and/or peroxidase activity. In a preferredembodiment, the additional enzyme is lipase.

[0068] The term “a step” of a process means at least one step, and itcould be one, two, three, four, five or even more process steps. Inother words the fatty acid oxidizing enzyme of the invention may beapplied in at least one process step, and the additional enzyme(s) mayalso be applied in at least one process step, which may be the same or adifferent process step as compared to the step where the fatty acidoxidizing enzyme is used.

[0069] The term “enzyme preparation” means a product containing at leastone fatty acid oxidizing enzyme. The enzyme preparation may alsocomprise enzymes having other enzyme activities, preferably lipolyticenzymes or enzymes having oxidoreductase activity, most preferablylipolytic enzymes. In addition to the enzymatic activity such apreparation preferably contains at least one adjuvant. Examples ofadjuvants, which are used in enzyme preparations for the paper and pulpindustry are buffers, polymers, surfactants and stabilizing agents.

[0070] Additional Enzymes

[0071] Any enzyme having protease, lipase, xylanase, cutinase,oxidoreductase, cellulase endoglucanase, amylase, mannanase, sterylesterase, and/or cholesterol esterase activity can be used as additionalenzymes in the use and process of the invention. Below some non-limitingexamples are listed of such additional enzymes. The enzymes written incapitals are commercial enzymes available from Novozymes A/S,Krogshoejvej 36, DK-2880 Bagsvaerd, Denmark. The activity of any ofthose additional enzymes can be analyzed using any method known in theart for the enzyme in question, including the methods mentioned in thereferences cited.

[0072] Examples of cutinases are those derived from Humicola insolens(U.S. Pat. No. 5,827,719); from a strain of Fusarium, e.g. F. roseumculmorum, or particularly F. solani pisi (WO 90/09446; WO 94/14964, WO94/03578). The cutinase may also be derived from a strain ofRhizoctonia, e.g. R. solani, or a strain of Alternaria, e.g. A.brassicicola (WO 94/03578), or variants thereof such as those describedin WO 00/34450, or WO 01/92502.

[0073] Examples of proteases are the ALCALASE, ESPERASE, SAVINASE,NEUTRASE and DURAZYM proteases. Other proteases are derived fromNocardiopsis, Aspergillus, Rhizopus, Bacillus alcalophilus, B. cereus,B. natto, B. vulgatus, B. mycoide, and subtilisins from Bacillus,especially proteases from the species Nocardiopsis sp. and Nocardiopsisdassonvillei such as those disclosed in WO 88/03947, and mutantsthereof, e.g. those disclosed in WO 91/00345 and EP 415296.

[0074] Examples of amylases are the BAN, AQUAZYM, TERMAMYL, and AQUAZYMUltra amylases. An example of a lipase is the RESINASE A2X lipase. Anexample of a xylanase is the PULPZYME HC hemicellulase. Examples ofendoglucanases are the NOVOZYM 613, 342, and 476 enzyme products.

[0075] Examples of mannanases are the Trichoderma reeseiendo-beta-mannanases described in Stahlbrand et al, J. Biotechnol. 29(1993), 229-242.

[0076] Examples of steryl esterases. peroxidases, laccases, andcholesterol esterases are disclosed in the references mentioned in thebackground art section hereof. Further examples of oxidoreductases arethe peroxidases and laccases disclosed in EP 730641; WO 01/98469; EP719337; EP 765394; EP 767836; EP 763115; and EP 788547. In the presentcontext, whenever an oxidoreductase enzyme is mentioned that requires orbenefits from the presence of acceptors (e.g. oxygen orhydrogenperoxide), enhancers, mediators and/or activators, suchcompounds should be considered to be included. Examples of enhancers andmediators are disclosed in EP 705327; WO 98/56899; EP 677102; EP 781328;and EP 707637. If desired a distinction could be made by defining anoxidoreductase enzyme system (e.g. a laccase, or a peroxidase enzymesystem) as the combination of the enzyme in question and its acceptor,and optionally also an enhancer and/or mediator for the enzyme inquestion.

[0077] These are particular embodiments of the present invention: Use ofa fatty acid oxidizing enzyme for reducing the deposition of pitch inthe paper making process. A process for reducing deposition of pitch inthe paper making process, wherein the process comprises treating thepulp and/or process water with an enzyme preparation comprising a fattyacid oxidizing enzyme; preferably a process wherein the pulp is amechanical pulp or a chemical pulp or a combination thereof; such as achemical pulp. The process as described above, wherein the enzyme isclassified in EC 1.13.11, preferably 1.13.11.12, preferably wherein theenzyme is derived from a strain of the genus Magnaporthaceae, preferablyM. salvinii or the genus Gaeumannomyces, preferably G. graminis. Theprocess described above, wherein the treatment is carried out by addinga substrate for the enzyme, preferably linoleic acid. The processdescribed above, wherein the enzyme preparation comprises a lipolyticenzyme and/or a further oxidoreductase. The process described abovewherein the treatment is carried out at a temperature is in the range20-90° C., preferably 40-70° C., and/or at a pH in the range 2-11,preferably 4-9.5, more preferably 6-9, and/or wherein treatment iscarried out in 10 minutes to 3 hours, preferably 15 minutes to 1 hour;and/or wherein the enzyme is added in a concentration in the range of0.0001-20 mg/g, preferably 0.0001-10 mg/g, more preferably 0.001-1 mg/gand most preferably 0.01-0.1 mg/g. In one embodiment of the aboveprocess the enzyme preparation is added in the storage chest or mixingchest before the paper machine.

[0078] The invention described and claimed herein is not to be limitedin scope by the specific embodiments herein disclosed, since theseembodiments are intended as illustrations of several aspects of theinvention. Any equivalent embodiments are intended to be within thescope of this invention. Indeed, various modifications of the inventionin addition to those shown and described herein will become apparent tothose skilled in the art from the foregoing description. Suchmodifications are also intended to fall within the scope of the appendedclaims. In the case of conflict, the present disclosure includingdefinitions will control.

[0079] Various references are cited herein, the disclosures of which areincorporated by reference in their entireties.

EXAMPLES Example 1

[0080] Measurement of the Activity of Fatty Acid Oxidizing Enzymes onLinoleic Acid

[0081] An “Oxi 3000 Oximeter” (WTW, Weilheim, Germany) with a TriOxmatic300 oxygen electrode and a standard reaction volume of 4 ml was used.

[0082] 10 mg linoleic acid (10 ml 60% linoleic acid) was dissolved in 1ml ethanol, and 2 micro liter Tween 20 was added. From this stocksubstrate solution 50 micro liter was added into a reaction beakercontaining 3.85 ml buffer solution (Britton-Robinson: 100 mM ofPhosphoric-, Acetic- and Boric acid; pH adjusted with NaOH) with a smallstir bar allowing the solution to be mixed well, and the oxygenelectrode was inserted into the reaction beaker. 100 micro literpurified enzyme solution was added, viz. (a) lipoxygenase derived fromMagnaporthe salvinii at a concentration of approx. 0.4 mg/ml; or (b)lipoxygenase derived from Gaeumannomyces. graminis at a concentration ofapprox. 0.76 mg/ml (which means approximately 0.02 mg/ml in the finalreaction). These lipoxygenases were prepared as previously described.The temperature was 25° C. The concentration of dissolved oxygen (mg/l)is measured and plotted as a function of time (min.). The enzymaticactivity is calculated as the slope of the linear part of the curve(mg/l/min.) after addition of the enzyme. The baseline was corrected bysubtraction when relevant, meaning that if the curve showing oxygenconcentration as a function of time had a slope of above about 0.05 mgoxygen/ml/min before addition of the fatty acid oxidizing enzyme (i.e.the control), this value was subtracted from the sample slope value.

[0083] Table 1 below shows the results of the experiments. TABLE 1 FattyAcid Oxidizing Enzyme (a) LOX from M. salvinii (b) LOX from G. graminispH mgO₂/ml/min mgO₂/ml/min 2 0.0 0.0 4 0.4 0.1 5 0.7 0.4 6 1.1 0.4 7 1.00.4 8 0.7 0.5 9 0.8 0.4 10  0.7 0.4 11  0.6 0.2

Example 2

[0084] Fatty Acid Oxidizing Enzymes

[0085] Four enzymes, viz. two laccases and two lipoxygenases were testedas described below. The laccase derived from Polyporus pinsitus had a MWby SDS-Page of 65 kDa, a pl by IEF of 3.5, and an optimum temperature atpH 5.5 of 60° C. The laccase derived from Coprinus cinereus had a MW bySDS-Page of 67-68 kDa, a pl by IEF of 3.5-3.8, and an optimumtemperature at pH 7.5 of 65° C. The enzymes were prepared and purifiedas described in WO 96/00290 and U.S. Pat. No. 6,008,029. The twolipoxygenases were derived from Magnaporthe salvinii and Gaeumannomycesgraminis, and they were prepared as described previously.

[0086] The enzyme dosage was adjusted to ensure maximum absorbancyincrease per minute at 234 nm /530 nm, viz. in the range of 0.1-0.25absorbancy units pr. min.

[0087] Substrate solution: 11.65 mg linoleic acid (60% Sigma), as wellas 12.5 ml 0.56 mM Syringaldazine (Sigma) in ethanol was mixed withdeionized water to a total volume of 25 ml.

[0088] 50 microliter of the enzyme preparation to be tested wastransferred to a quartz cuvette containing 900 microliter phosphatebuffer (50 mM, pH 7.0) and 50 microliter of the substrate solution Thecuvette was placed in a spectrofotometer, thermostated at 23° C., andthe absorbancies at 234 nm and 530 nm were measured as a function oftime. The absorbancy at 530 nm is indicative of degradation ofsyringaldazine, whereas the absorbancy at 234 nm is indicative ofdegradation of linoleic acid. The absorbancy increase as a function oftime is calculated on the basis of minutes 2 to 4 of the reaction time,i.e. d(A₂₃₄)/dt, as well as d(A₅₃₀)/dt.

[0089] The results are shown in Table 2 below. Of these four enzymes,only the two lipoxygenases qualify as a fatty acid oxidizing enzyme asdefined herein. This is because RRD=Reaction RateDifference=(dA₂₃₄/dt−dA₅₃₀/dt) is above zero only for these two enzymes.TABLE 2 dA₅₃₀/dt dA₂₃₄/dt dA₂₃₄/dt − dA₅₃₀/dt Enzyme (units/min)(units/min) (units/min) Polyporus pinsitus 0.20 0.002* −0.20 laccaseMagnaporthe salvinii 0.0001* 0.13 0.13 lipoxygenase Coprinus cinereus0.17 −0.001* −0.17 laccase Gaeumannomyces −0.03* 0.21 0.21 graminislipoxygenase

Example 3

[0090] Reduction of Pitch with a Fatty Acid Oxidizing Enzyme

[0091] A model pitch is prepared as follows:

[0092] 50% Linoleic Acid 60% (Sigma L-1626).

[0093] 20% Abietic Acid (Sigma A9424).

[0094] 20% Oleic Acid (Merck 471).

[0095] 5% Cholesterol-Linoleate (Sigma C-0289).

[0096] 5% Olive Oil (Sigma 0-1500).

[0097] Mixed for 30 minutes at 65° C. Stored in refrigerator for nolonger than 30 days.

[0098] Preparation of 0.1% pitch suspension:

[0099] 50 mg model pitch

[0100] 1 ml ethanol.

[0101] 1 ml 0.1 M NaOH.

[0102] 48 ml buffer (50 mM borate pH 9.0) Mixed for 10 minutes at roomtemperature.

[0103] Circular paper pieces (diameter=5.5-6 mm; Multicopy 80 g/m²) aretransferred to the wells of two 96-well microtiterplates (ID 269620 fromNUNC) designated A and B. Two other similar microtiterplates C and D arealso used, but without paper pieces. 100 microliter of the 0.1% pitchsuspension is added into each of the wells of each of these fourmicrotiterplates. A lipoxygenase derived from Magnaporthe salvinii asdescribed previously is used as the fatty acid oxidizing enzyme, and itis added to the wells of microtiterplates A and C to obtain anin-well-concentration of 10 ppm. A similar amount of buffer (50 mMborate pH 9.0) is added to the wells of microtiterplates B and D. Themicrotiterplates are then incubated during shaking (600 rpm) for 30minutes. After 30 minutes, 20 microliter of the enzyme-treated pitchsuspension is transferred onto a second set of microtiterplatescorresponding to microtiterplates A-D (Corning Inc. Costar UV plate 96well No. 3635) each containing 200 microliter ethanol per well(solubilizing the pitch components). Abietic acid, a major component ofthe pitch, absorps strongly at about 255 nm. Accordingly, A₂₅₅ isindicative of the amount of pitch remaining in the suspension. A₂₅₅ isdetermined as the average of 8 identical experiments, and the amount ofpitch adsorbed onto the paper is estimated based on the variation inA₂₅₅ measured in the pitch suspensions obtained after incubation withand without paper present (after 11× dilution in ethanol).

[0104] The results are shown in a table like the below Table 3. Thebasic (blind) adsorption of pitch onto the paper in the absence of afatty acid oxidizing enzyme may be calculated as the ratio D/B. Theeffect of the enzyme (the sample) as regards the adsorption of pitch tothe paper may be calculated as the ratio C/A. One way of showing hat theenzyme has caused a reduction in the deposition of pitch is if (C/A−D/B)is below zero. Alternatively, the enzyme effect may be calculated as((C−A)−(D−B)), and if this value is below zero, this would be anotherway of showing the effect of the enzyme on the deposition of pitch.Other solid materials than paper may also be tested, e.g. metal, andtextile (Style 400 cotton). The above ways of showing reduction in pitchdeposition are applicable by analogy as regards deposition on the othersolid materials.

[0105] Of course, the assay-pH (i.e. buffer), and the assay-temperatureis selected paying regard to the characteristics of the fatty acidoxidizing enzyme in question, e.g. an assay pH of around 4, 5, 6, 7, 8,9, 10, or 11; and an assay-temperature of around 10, 15, 20, 25, 30, 37,40, 50, 60, 70, 80, 90 or 95° C. TABLE 3 With M. salvinii Without M.salvinii A₂₅₅ lipoxygenase lipoxygenase With paper A B Without paper C D

Example 4

[0106] Bleaching Paper with a Fatty Acid Oxidizing Enzyme

[0107] Unbleached Kraft Pulp derived from Eucalyptus grandis was used.The pulp was repulped at 4% consistency in a pulper manufactured byLoretzen and Wettre. Repulping was done in buffer (Britton-Robinson) atpH=9.0.

[0108] Britton Robinson buffer:

[0109] 100 mM Phosphoric acid (85%) 6.28 ml

[0110] 100 mM Acetic acid (100%) 5.72 ml

[0111] 100 mM Boric acid 6.18 g

[0112] Dem. water up to 1000 ml

[0113] The pH was adjusted to 9.0 by addition of sodium hydroxide.

[0114] After repulping the pulp slurry was diluted to 1% consistency byaddition of buffer and pH was readjusted to pH =9.0.

[0115] Treatments with a fatty acid oxidizing enzyme were carried out inbeakers containing 3 g dry pulp i.e. 300 ml pulp slurry. The treatmentswere carried out at 25° C. in a water bath with agitation by magneticstir bars, 500 rpm. 333 ppm linoleic acid was added to all beakers. Thefatty acid oxidizing enzyme used was a purified lipoxygenase derivedfrom Gaeumannomyces graminis prepared as described previously. Theamount of enzyme used appears from Tables 4 and 5 below. The enzymetreatment was carried out for 2 hours. Two beakers were run for eachcondition.

[0116] After two hours the enzyme reaction was stopped by addition of 5ml (fixed amount) of NaOH (27.65% solution), this raises pH to >12, anddeactivates the enzyme.

[0117] The content of the beaker was transferred quantitatively to a1000 ml beaker using 700 ml deionised water. This pulp suspension waspoured onto a Buchner funnel (15 cm diameter) with a filter paper. Apaper sheet was formed by sucking the water out. The paper sheet wasremoved from the funnel and separated from the filter paper. The sheetwas pressed in a sheet press manufactured by Lorentzen and Wettre. Thesheet was pressed in a sandwich of metal plate, 2 blotting papers, 2filter papers, the sheet, 2 filter papers, 2 blotting papers, metalplate at 0,4 MPa for 5.5 min. Wet papers were replaced by dry ones andthe pressing repeated at 0.4 MPa for 2 min. The sheets were air driedovernight.

[0118] The brightness of the sheets was measured using a MacbethColor-Eye 7000 reflectometer. The brightness was recorded at 600nm. 4measurements were done at each sheet. The results obtained are shown inTable 4 below.

[0119] The Kappa Number, which describes the degree of delignificationof a pulp, was also determined for each sheet using the method describedin Tappi Test Methods T236 (Tappi Press). The amounts used for eachdetermination was ¼ of that described in the standard method. The drymatter content of the sheets was determined to calculate the Kappa no.The results obtained are shown in Table 5 below. TABLE 4 BrightnessBrightness Reflection at Reflection at 600 nm 600 nm Brightness Sheet 1Sheet 2 Reflection at Average of 4 Average of 4 600 nm LOX [mg/l]determinations Determinations Average 0 46.93 46.60 46.77 0.1 49.6950.23 49.96 1.3 48.51 47.15 47.83 3.2 49.16 50.12 49.64 6.3 48.56 52.5650.56

[0120] TABLE 5 Kappa No. Kappa No. Sheet 1 Sheet 2 Average of 3 Averageof 3 Kappa No. LOX [mg/l] determinations determinations Average 0 18.4418.43 18.44 0.1 14.80 14.68 14.74 1.3 14.94 14.93 14.94 3.2 14.65 14.4214.54 6.3 14.83 14.40 14.61

Example 5

[0121] De-Inking Old Newsprint with a Fatty Acid Oxidizing Enzyme

[0122] 200 g shredded old newsprint was placed in a Hobart Mixertogether with 1500 ml of water. The water bath temperature was set at45° C. Mixing occurred at low speed for about 0.5-1 minutes. Then 3.6kg/ton (7 lb/ton) of surfactant and 2 mg (10 mg/ton pulp) of alipoxygenase derived from Magnaporthe salvinii prepared as describedpreviously was added to the mixer, following which 500 ml of water wasadded to Hobart and mixed well. The mixer was run on low speed for 30minutes. The pulper temperature was set at 45° C., and the pH at 7.

[0123] Half of the pulp was transferred from the mixer to a containerand diluted to 10 l. Stirring took place for 2 minutes.

[0124] Feed pads: 30 ml of pulp was measured from the mixer and dilutedto 300 ml with water and mixed well. The pulp was filtered through aWattman#40 filter paper under vacuum. The pad was dried at 90° C. (195°F.) for 10 minutes.

[0125] Regular washing of pads: 900 ml of pulp slurry was measured fromthe container, poured slowly onto an 80 mesh sieve and shaken slowlyuntil all free water drained. All pulp was removed and put into a 1000ml beaker, which was filled with water up to the 900 ml line. The pulpwas slowly stirred. 300 ml of the pulp slurry was measured and filteredthrough a Wattman#40 filter paper under vacuum. The pad was dried at 90°C. (195° F.) for 10 minutes.

[0126] Hyper-washing of pads: 900 ml of pulp slurry was measured fromthe container, poured slowly onto an 80 mesh sieve and shaken slowlyuntil all free water drained. The pulp was rinsed with faucet water for3 minutes, removed and put into a 1000 ml beaker, which was filled withwater up to the 900 ml line. The pulp was slowly stirred. 300 ml of thepulp slurry was measured and filtered through a Wattman #40 filter paperto make a filter pad. The pads were dried on a speed dryer at 90° C.(195° F.) for 10 minutes.

[0127] The brightness of the pads was determined by a Macbeth color eyeusing a Tappi standard method (T452).

[0128] Comparative experiments were conducted as described above withtwo commercial enzymes, viz. the lipase RESINASE A 2×, and the cellulaseDENIMAX L, both commercially available from Novozymes A/S, Krogshoejvej36, DK-2880 Bagsvaerd, Denmark. These enzyme preparations were used inan amount of 0.51 kg per ton pulp (1 lb/t).

[0129] The results are shown in Table 6 below. TABLE 6 Brightness EnzymeFeed Pads Washed Pads Hyper-washed Pads Control (no enzyme) 38.5 41.745.9 Fatty acid oxidizing 38.1 44.0 48.2 enzyme RESINASE A 2X 40.6 42.945.9 (lipase) DENIMAX L 37.6 39.0 44.2 (cellulase)

1. A process for manufacturing a paper material, which process comprisesthe step of treating a papermaking pulp and/or process water with afatty acid oxidizing enzyme.
 2. The process of claim 1, furthercomprising the steps of forming and drying the enzyme-treated pulp. 3.The process of claims 1, wherein the enzyme-treatment results in reduceddeposition of pitch.
 4. The process of claim 1, wherein theenzyme-treatment results in bleaching of the paper material.
 5. Theprocess of any one of claims 1-4, in which the papermaking pulpcomprises a chemical pulp.
 6. The process of any one of claims 1-4,wherein the papermaking pulp comprises pulp from recycled printed papermaterial.
 7. The process of claim 6, wherein the enzyme-treatmentresults in bleaching of the paper material.
 8. The process of claim 6,wherein the enzyme-treatment results in de-inking of the paper material.9. The process of any one of claims 1-4, wherein a substrate for theenzyme is added before or during the enzyme-treatment step.
 10. Theprocess of any one of claims 1-9, further comprising treating thepapermaking pulp and/or process water with an additional enzyme selectedfrom the group consisting of a lipase, cutinase, oxidoreductase,cellulase, amylase, mannanase, steryl esterase, cholesterol esteraseactivity, and combinations thereof. 11 The process of claim 10, whereinthe oxidoreductase enzyme has laccase activity, peroxidase activity orboth laccase and peroxidase activity.
 12. The process of claim 10,wherein the additional enzyme has lipase activity.
 13. The process ofany one of claims 10-12, wherein the treatment with the additionalenzyme occurs before, concomitantly with, and/or after the treatmentwith the fatty acid oxidizing enzyme.
 14. The process of any one ofclaims 1-13, wherein the fatty acid oxidizing enzyme is a lipoxygenase.15. A process for reducing pitch deposition, which process comprises thestep of treating a papermaking pulp and/or process water with a fattyacid oxidizing enzyme, wherein the enzyme-treatment results in reduceddeposition of pitch.
 16. The process of claim 15, wherein the fatty acidoxidizing enzyme is a lipoxygenase.
 17. A process for bleaching a papermaterial, comprising the step of treating a papermaking pulp and/orprocess water with a fatty acid oxidizing enzyme, wherein theenzyme-treatment results in bleaching of the paper material.
 18. Theprocess of claim 17, wherein the fatty acid oxidizing enzyme is alipoxygenase.
 19. A process for de-inking a printed paper material,which process comprises the step of treating a papermaking pulp and/orprocess water with a fatty acid oxidizing enzyme, wherein theenzyme-treatment results in de-inking of the paper material.
 20. Theprocess of claim 19, wherein the fatty acid oxidizing enzyme is alipoxygenase.